title: Purification and gene cloning of alpha-methylserine aldolase from Ralstonia sp. strain AJ110405 and application of the enzyme in the synthesis of alpha-methyl-L-serine.
abstract: By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.
authors.affiliation: Aminoscience Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. hiroyuki_nozaki@ajinomoto.com
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nozaki
authors.forename: Shinji
authors.initials: S
authors.lastname: Kuroda
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Watanabe
authors.forename: Kenzo
authors.initials: K
authors.lastname: Yokozeki
mesh_terms: D000409:Alanine / Q000378:metabolism; D027703:Amaranthaceae / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D042621:Comamonadaceae / Q000201:enzymology; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005557:Formaldehyde / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D021122:Protein Subunits; D043367:Ralstonia / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D005557:Formaldehyde; D012694:Serine; D005634:Fructose-Bisphosphate Aldolase; D000409:Alanine